Crystal structures of main proteases of SARS-CoV-2 variants bound to a benzothiazole-based inhibitor

Main protease (M pro) serves as an indispensable factor in the life cycle of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as its constantly emerging variants and is therefore considered an attractive target for antiviral drug development. Benzothiazole-based inhibitors targeting M pro have recently been investigated by several groups and proven to be promising leads for coronaviral drug development. In the present study, we determine the crystal structures of a benzothiazole-based inhibitor, YH-53, bound to M pro mutants from SARS-CoV-2 variants of concern (VOCs) or variants of interest (VOIs), including K90R (Beta, B.1.351), G15S (Lambda, C.37), Y54C (Delta, AY.4), M49I (Omicron, BA.5) and P132H (Omicron, B.1.1.529). The structures show that the benzothiazole group in YH-53 forms a C-S covalent bond with the sulfur atom of catalytic residue Cys145 in SARS-CoV-2 M pro mutants. Structural analysis reveals the key molecular determinants necessary for interaction and illustrates the binding mode of YH-53 to these mutant M pros. In conclusion, structural insights from this study offer more information to develop benzothiazole-based drugs that are broader spectrum, more effective and safer.


Introduction
The outbreak of coronavirus disease-19  in 2019 turned into a global pandemic and seriously impacted the public health system and economy [1,2]. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was identified as the causative agent soon after the COVID-19 outbreak [3]. This agent belongs to the βcoronavirus genus, which also includes SARS-CoV and MERS-CoV [4,5]. Before the SARS-CoV-2 outbreak, SARS-CoV and MERS-CoV caused the most recent two other coronavirus outbreaks that occurred in China in 2003 [6] and in Saudi Arabia in 2012 [7], respectively. Unlike the extinguished outbreak of SARS-CoV and MERS-CoV, the SARS-CoV-2 outbreak has lasted for three years and has caused 753 million cases of COVID-19 as of January 31, 2023, including 6.8 million deaths (https://covid19.who.int/).
To develop an antiviral drug against SARS-CoV-2, a series of studies evaluated the potential drug targets of SARS-CoV-2. Among them, the main protease (M pro ) is one of the foremost appealing targets in the life cycle and pathogenicity of the virus [11][12][13]. SARS-CoV-2 M pro is an indispensable enzyme in viral replication and transcription [14], and it is extremely critical for the cleavage of two polypeptide (pp1a and pp1ab) sequences after a glutamine residue [13]. It has a unique feature of substrate recognition that is not shared by human proteases [13,15,16]. Moreover, M pro is highly conserved in SARS-CoV-2, which also makes it possible to develop broad-spectrum coronavirus drugs [17][18][19]. In summary, M pro is an important and conserved target for the inhibition of SARS-CoV-2. Inhibitors targeting M pro can effectively prevent the replication of SARS-CoV-2 and therefore represent promising drug candidates.
Over a sustained period, particularly from the start of the pandemic, drug repurposing has been a successful alternative to drug development [20,21]. Therefore, previously effective small molecule inhibitors against M pro are potential pools to combat the coronavirus disease COVID-19 [22][23][24][25]. Among many researched inhibitors, the benzothiazole-based small molecule YH-53 shows exciting prospects [26]. YH-53 is a peptidomimetic inhibitor that effectively inhibits the action of M pro in SARS-CoV-2 in enzymatic and cellular antiviral assays [26][27][28][29]. YH-53 offers a number of advantages, including good metabolic and excretion profile, good pharmacokinetics and no apparent toxicity [26,28]. As a covalent inhibitor, YH-53 primarily utilizes the benzothiazole moiety as a warhead, and it is derived from a tetrapeptide inhibitor by the addition of a trifluoromethyl ketone fraction [26]. The benzothiazole group (electron attracting group) can be greatly enhanced to form covalent bonds with the M pro active site, temporarily inactivating the enzyme and completely blocking the replication of SARS-CoV-2 [26,29]. These studies revealed that YH-53 can establish a covalent bond with the active site of M pro , which suggests that it has the potential as a lead candidate for further drug development [26]. Our previous studies mainly focused on the enzymatic inhibition of YH-53 against M pro s from different coronaviruses, including SARS-CoV, MERS-CoV and SARS-CoV-2, and illustrated the structural basis for the inhibition [29].
In the present study, we investigated the details of the interaction between the benzothiazole-based small molecule YH-53 and the mutant M pro s, each carrying a single mutation, by solving the complex structures. These mutations come from different SARS-CoV-2 variants: B.1.351 Beta (K90R), C.37 Lambda (G15S), Delta AY.4 (Y54C), BA.5 Omicron (M49I) and B.1.1.529 Omicron (P132H). The results provided structural insights for understanding the precise inhibition mechanism of the inhibitor against different M pro s and strongly indicated the potential of YH-53 to be developed as a broad-spectrum drug candidate. Additionally, the present study will ultimately provide theoretical guidance for finding alternatives to treat different variants of SARS-CoV-2.

Materials and Methods
Expression and purification of M pro mutants from human SARS-CoV-2 A plasmid containing the gene encoding wild-type SARS-CoV-2 M pro was constructed based on a previous method [29]. Using the wildtype plasmid as a template, site-directed mutagenesis was performed to form each of the variations (K90R, G15S, Y54C, M49I and P132H). Nucleotide sequences were confirmed by DNA sequencing. The plasmids containing genes encoding mutant M pro s were introduced into competent cells E. coli Rosetta DE3 for protein expression. The expression and purification of the M pro mutants with His-tag were performed according to the method described previously [18]. Furthermore, TEV protease was used to remove the N-terminal His-tag.

Crystallization of various M pro mutants in complex with YH-53
Five mutants of SARS-CoV-2 M pro were concentrated to 5 mg/mL, and incubated on ice with YH-53 at a molar ratio of 1:3 for 30 min. Crystallization was performed at 18°C using the hanging drop vapour-diffusion method.

Data collection, structure determination, and refinement
The crystals were stabilized and cryoprotected by the addition of a reservoir solution containing 20% glycerol and then flash cooled in liquid nitrogen. All X-ray diffraction data sets were collected at 100 K at BL10U2 of the Shanghai Synchrotron Radiation Facility (Shanghai, China). Diffraction data were autoprocessed using the aquarium pipeline 24 [30], and the data processing statistics are listed in Table 1. All the structures of M pro mutants in complex with YH-53 were determined by the molecular replacement method using the program Phaser [31]. The maximum likelihood-based refinement of the atomic positions and temperature factors was performed with Phenix [32]. The atomic model was fit with the program Coot [33]. The stereochemical quality of the final model was assessed with MolProbity [34]. The structural refinement statistics of the mutant M pro s from SARS-CoV-2 variants in complex with YH-53 are shown in Table 1

Expression and purification of M pro mutants
All five mutant M pro s were expressed in E. coli cells and purified to homogeneity by gel filtration. The elution volume and molecular weight (approximately 35 kDa) of each M pro mutant were almost identical (Figure 1), which was also observed for wild-type M pro .

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Crystal structures of M pro mutants bound to YH-53 These data indicated that these mutant M pro s share similar protein folding efficiency with the wild-type M pro .

Crystal structures of YH-53 in complex with M pro mutants from SARS-CoV-2 variants
We then determined the crystal structures of M pro mutants of SARS-CoV-2 in complex with YH-53 using the cocrystallization method.  Figure 2).

Comparison of the binding modes of YH-53 with different M pro mutants
To better understand the binding mode of YH-53 with different mutants, we extracted the electron density map of YH-53 and the catalytic residue Cys145 (Figure 2). The electron density diagram showed that the benzothiazole group of YH-53 is close to the sulfur atom of the catalytic residue Cys145 in the mutant SARS-CoV-2 M pro , which promotes the formation of the covalent bond ( Figure 3). Cys145 is a nucleophilic reagent in the hydrolysis process of SARS-CoV-2 M pro mutants, and the binding of YH-53 inhibits their hydrolysis reaction. The inhibitor binding sites (catalytic active sites) of the five mutants were superposed to compare the structural differences caused by the M pro mutation when bound to YH-53. The result shows that there are slight differences in the orientation of YH-53 in the substrate-binding sites of different M pro mutants ( Figure 3).

Discussion
In the past three years, the COVID-19 pandemic has continued, causing serious social, economic, and political chaos. Like other RNA viruses, SARS-CoV-2 is constantly changing through mutation, and each virus with a unique sequence is considered to be a new variant. It is an urgent task for the scientific and pharmaceutical communities to develop effective drugs or therapies against SARS-CoV-2 and its variants. Among several structural and nonstructural SARS-CoV-2 proteins, M pro has been designated as a potential therapeutic target for drug development. Inhibiting M pro will prevent virus replication and constitutes one of the potential anticoronavirus strategies. In recent years, a large number of studies have focused on the use of old drugs as a reasonable treatment for COVID-19. The success of drug reuse has attracted extensive research interest. Here, we evaluated the details of the interactions between the benzothiazole-based small molecule YH-53 and five different M pro mutants. These mutations exist in different SARS-CoV-2 variants: B. The crystal structures of different M pro mutants of SARS-CoV-2 bound to YH-53 were analyzed. In terms of overall structure, no significant changes in the conformation of the five M pro mutants when bound to YH-53 were observed. As indicated by the electron density map of YH-53 and the catalytic residue Cys145, there was little difference in the orientation of the benzothiazole group of YH-

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Crystal structures of M pro mutants bound to YH-53 53. We marked the amino acids interacting with YH-53 in different mutants with the stick pattern and found that although the residues contributing to contacts with YH-53 in different M pro mutants were slightly different, the catalytic dyads (Cys145 and His41) in all mutants were involved in the interaction. As it is believed that the molecules that interact strongly with these catalytic residues are the key to establishing strong binding with and inhibition against this enzyme, the present results thus indicate that YH-53 may retain potent inhibition against these SARS-CoV-2 M pro variants. The continuous emergence of SARS-CoV-2 variants has seriously

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Crystal structures of M pro mutants bound to YH-53 endangered public health safety. Continuous research on inhibitors is of great benefit to quickly understand their current and future antiviral effects. Our research showed that the binding mode of YH-53 is not affected by the tested mutants, indicating that YH-53 may retain inhibitory efficacy against M pro mutants. Therefore, the present study provides a theoretical basis for the treatment of SARS-CoV-2 variants and further drug development.